Showing papers in "Microscopy and Microanalysis in 2003"
TL;DR: The application of a new automated, unbiased, multivariate statistical analysis technique to very large X-ray spectral image data sets, based in part on principal components analysis, returns physically accurate component spectra and images in a few minutes on a standard personal computer.
Abstract: Spectral imaging in the scanning electron microscope (SEM) equipped with an energy-dispersive X-ray (EDX) analyzer has the potential to be a powerful tool for chemical phase identification, but the large data sets have, in the past, proved too large to efficiently analyze. In the present work, we describe the application of a new automated, unbiased, multivariate statistical analysis technique to very large X-ray spectral image data sets. The method, based in part on principal components analysis, returns physically accurate (all positive) component spectra and images in a few minutes on a standard personal computer. The efficacy of the technique for microanalysis is illustrated by the analysis of complex multi-phase materials, particulates, a diffusion couple, and a single-pixel-detection problem.
306 citations
TL;DR: In this article, a new approach based on prediction of specimen position during the tilt series from the position at previous tilts is proposed, which achieves both the speed of pre-calibration and the reliability of the traditional approach.
Abstract: The traditional approach to automated tilt series acquisition involves tracking specimen position and measuring focus after every tilt, sometimes with time-consuming magnification changes [1]. The predictable performance of modern goniometers has allowed the development of a different method in which the translational specimen holder movements are precalibrated with a rapid, coarse tilt series, then the full tilt series is acquired relying on this calibration [2]. Although this method is efficient, with thick specimens imaged at 300 KV in our laboratory we have found it susceptible to drift, the less accurate adjustment of eucentric height for cryo-samples, and other non-ideal features of the specimen or microscope. I thus developed a program, SerialEM, that uses a new approach based on prediction of specimen position during the tilt series from the position at previous tilts. This method achieves both the speed of pre-calibration and the reliability of the traditional approach.
254 citations
TL;DR: In this paper, the authors present a tool for the allocation of organic and inorganic components in calcified biominerals called Confocal Micro-Raman Spectroscopy (CMS).
Abstract: Part I: Theory.- Confocal Microscopy.- Introduction into the Fundamentals of Raman Spectroscopy.- Raman Equipment.- Software Requirements and Data Analysis in Confocal Raman Microscopy.- Part II: Applications.- Identification of Pathogens.- Imaging of Individual Carbon Nanotubes and Isolated Graphene Sheets.- Raman Microscopy in Geosciences.- Raman Microscopic Imaging of Cells and Applications Monitoring the Uptake of Drug Delivery Systems.- Confocal Micro-Raman Spectroscopy: A Tool for the Allocation of Organic and Inorganic Components in Calcified Biominerals.- Pharmaceutical Applications of Confocal Raman Microscopy.- Applications of Confocal Raman Microscopy in Pharmaceutical Development.- Characterization of Therapeutic Coatings on Medical Devices.- Raman Imaging of Plant Cell Walls.- Confocal Raman Imaging of Polymeric Materials.- Stress Measurement by Means of Raman Microscopy
238 citations
TL;DR: The fundamental ion/solid interactions that govern the FIB milling process are examined and discussed with respect to the preparation of electron transparent membranes and the roles of incident ion attack angle, beam current, trench geometry, raster pattern, and target-material-dependent removal rates are considered.
Abstract: The focused ion beam (FIB) tool has been successfully used as both a stand alone analytical instrument and a means to prepare specimens for subsequent analysis by SEM, TEM, SIMS, XPS, and AUGER. In this work, special emphasis is given to TEM specimen preparation by the FIB lift-out technique. The fundamental ion/solid interactions that govern the FIB milling process are examined and discussed with respect to the preparation of electron transparent membranes. TRIM, a Monte Carlo simulation code, is used to physically model variables that influence FIB sputtering behavior. The results of such computer generated models are compared with empirical observations in a number of materials processed with an FEI 611 FIB workstation. The roles of incident ion attack angle, beam current, trench geometry, raster pattern, and target-material-dependent removal rates are considered. These interrelationships are used to explain observed phenomena and predict expected milling behaviors, thus increasing the potential for the FIB to be used more efficiently with reproducible results.
100 citations
TL;DR: It is shown that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy and evidence is presented that this pronuclear accumulation may be positively correlated with development to the blastocyst stage—in the same embryo—thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential.
Abstract: We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.
86 citations
TL;DR: An improved algorithm for adaptive 3-D skeletonization of noisy images enables the tracing of dye-injected neurons imaging by fluorescence confocal microscopy and HRP-stained neurons imaged by transmitted-light brightfield microscopy.
Abstract: Automated three-dimensional (3-D) image analysis methods are presented for tracing of dye-injected neurons imaged by fluorescence confocal microscopy and HRP-stained neurons imaged by transmitted-light brightfield microscopy. An improved algorithm for adaptive 3-D skeletonization of noisy images enables the tracing. This algorithm operates by performing connectivity testing over large N × N × N voxel neighborhoods exploiting the sparseness of the structures of interest, robust surface detection that improves upon classical vacant neighbor schemes, improved handling of process ends or tips based on shape collapse prevention, and thickness-adaptive thinning. The confocal image stacks were skeletonized directly. The brightfield stacks required 3-D deconvolution. The results of skeletonization were analyzed to extract a graph representation. Topological and metric analyses can be carried out using this representation. A semiautomatic method was developed for reconnection of dendritic fragments that are disconnected due to insufficient dye penetration, an imaging deficiency, or skeletonization errors.
85 citations
TL;DR: This innovative technique offers several advantages over current biomechanics methodologies, including simultaneous visualization of scaffold and/or cell microstructure in three dimensions during mechanical loading; quantification of macroscopic mechanical parameters including true stress and strain; and the ability to perform multiple analyses on the same specimen.
Abstract: At present, mechanisms by which specific structural and mechanical properties of the three-dimensional extracellular matrix microenvironment influence cell behavior are not known. Lack of such knowledge precludes formulation of engineered scaffolds or tissue constructs that would deliver specific growth-inductive signals required for improved tissue restoration. This article describes a new mechanical loading–imaging technique that allows investigations of structural–mechanical properties of biomaterials as well as the structural–mechanical basis of cell–scaffold interactions at a microscopic level and in three dimensions. The technique is based upon the integration of a modified, miniature mechanical loading instrument with a confocal microscope. Confocal microscopy is conducted in a reflection and/or fluorescence mode for selective visualization of load-induced changes to the scaffold and any resident cells, while maintaining each specimen in a “live,” fully hydrated state. This innovative technique offers several advantages over current biomechanics methodologies, including simultaneous visualization of scaffold and/or cell microstructure in three dimensions during mechanical loading; quantification of macroscopic mechanical parameters including true stress and strain; and the ability to perform multiple analyses on the same specimen. This technique was used to determine the structural–mechanical properties of three very different biological materials: a reconstituted collagen matrix, a tissue-derived biomaterial, and a tissue construct representing cells and matrix.
74 citations
TL;DR: It is not easy to understand how the electron microscopes and electron microscope techniques that the authors know today developed from the primitive ideas of the first microscopists of the 1930s, but a guide to the principal stages in the development of the subject and to the main actors and their publications are available.
Abstract: It is not easy to understand how the electron microscopes and electron microscope techniques that we know today developed from the primitive ideas of the first microscopists of the 1930s. Newcomers to the subject in particular, their time almost fully occupied with grasping practical methods and modern computing techniques, can rarely devote much attention to the history of their subject. For some, however, this is a source of frustration: If a guide to the principal stages in the development of the subject and to the main actors and their publications were available, they would find the time to study it.
71 citations
TL;DR: This presentation describes my personal attempt to setup such a general software package for TEM/STEM use using java as the unique programming language.
Abstract: It is surprising that manufacturers of TEM and STEM microscopes do not provide suitable user-friendly software packages for the analysis and simulation of diffraction patterns, twoand many-beams conventional TEM images, high resolution micrographs, etc. Nothing close to the software tools available for example in X-Ray crystallography has yet been developed. This presentation describes my personal attempt to setup such a general software package for TEM/STEM use. The present development has been started in 1999 using java as the unique programming language. Java is an object oriented language that has been long considered as a low performing computer language not adapted to scientific calculations. This is no more the case as the execution speed of well written Java code is comparable to that of “C”. At present major scientific software, for example Mathematica, are either written in Java language or offer a link to Java written code.
65 citations
TL;DR: It is shown that the technique used to make thin specimens for electron microscopy affects the structure of the metallic glass, with ion thinning inducing more structural order than electropolishing.
Abstract: We have used fluctuation microscopy to reveal the presence of structural order on length scales of 1-2 nm in metallic glasses. We compare results of fluctuation microscopy measurements with high resolution transmission electron microscopy and electron diffraction observations on a series of metallic glass samples with differing degrees of structural order. The agreement between the fluctuation microscopy results and those of the other techniques is good. In particular, we show that the technique used to make thin specimens for electron microscopy affects the structure of the metallic glass, with ion thinning inducing more structural order than electro-polishing. We also show that relatively minor changes in the composition of the alloy can have a significant effect on the medium-range order; this increased order is correlated with changes in mechanical behavior.
61 citations
TL;DR: It is concluded that the image contrast, signal, and signal-to-noise ratio (SNR) are key to the achievable reconstruction quality and, as such, the technique may be of limited value for high energy loss/small inelastic cross section edges.
Abstract: The length scales on which materials microstructures are being formed, grown, and even designed are becoming increasingly small and increasingly three-dimensional. For such complex structures two-dimensional transmission electron microscopy (TEM) analysis is often inadequate and occasionally misleading. One approach to this problem is the modification of electron tomography techniques, developed for structural biology, for use in materials science. Energy-Filtered (EF) TEM elemental distribution images approximate to true projections of structure, and, as such, can be used to reconstruct the three-dimensional distribution of chemical species. A sample holder has been modified to allow the high tilt (+/-60 degrees ) required for tomography and a semiautomatic acquisition script designed to manage energy-loss acquisition. Tilt series data sets have been acquired from two widely different experimental systems, Cr carbides in 316 stainless steel and magnetite nanocrystals in magnetotactic bacteria, demonstrating single- and multiple-element tomography. It is shown that both elemental maps and jump-ratio images are suitable for reconstruction, despite the effects of diffraction contrast in the former and thickness changes in the latter. It is concluded that the image contrast, signal, and signal-to-noise ratio (SNR) are key to the achievable reconstruction quality and, as such, the technique may be of limited value for high energy loss/small inelastic cross section edges.
TL;DR: In this paper, the authors proposed a reproducible method for film thickness determination using high-resolution transmission electron microscopy (HR-TEM) and beam localization for silicon oxynitride films.
Abstract: High-resolution transmission electron microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by nonspecialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark-field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods has been steadily improved reaching now into the sub-Angstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this article, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this article is the proposal of a reproducible method for film thickness determination.
TL;DR: The simultaneous change of Φ and SEE in the case of oxygen covered tungsten have been pointed out and a direct relationship between them can be supposed.
Abstract: The changes of the work function (Phi) and the secondary electron emission (SEE) of oxygen covered polycrystalline tungsten occurring after ion sputtering and heat treatments have been investigated. The chemical composition was analyzed by X-ray photoelectron spectroscopy (XPS), and the electron emission properties by work function spectroscopy (WFS). We observed in what manner the chemical changes of the surface are reflected in the work function and SEE. The simultaneous change of Phi and SEE in the case of oxygen covered tungsten have been pointed out and a direct relationship between them can be supposed.
TL;DR: The scanning electron microscope (SEM) as discussed by the authors is a member of the same family of imaging devices as FAX machines and televisions, which is used to collect and amplify the signals emitted by a focussed beam of electrons, typically in the range from a few hundred eV to about 30 keV.
Abstract: The scanning electron microscope (SEM), a member of the same family of imaging devices as FAX machines and televisions. is the most widely used of all electron beam tools with some 40,000 units distributed worldwide. The SEM employs a focussed beam of electrons, with energies typically in the range from a few hundred eV to about 30 keV, which is rastered across the surface of a sample in a rectangular scan pattern. Signals emitted under this electron irradiation are collected, amplified, and then used to modulate the brightness of a suitable display device which is being scanned in synchronism with probe beam. This arrangement has several important benefits; (1) the magnification, which is the ratio of the areas scanned on the display device and on the sample, is obtained geometrically and so does not rely on lenses; (2) any emission from the sample that can be collected can be used to form an image; (3) multiple images using different signals can be collected simultaneously; (4) because the signal is acquired sequentially it can be processed and enhanced before it is displayed. The resolution of the SEM cannot be better than the dimension of the pixels used to display the image, and this limitation controls the imaging performance for all magnifications lower than about 20,000x. At higher magnifications, however, the resolution may be determined by other considerations including the type of signal that is being employed, the signal to noise ratio, the physical size of the electron probe, and the width of the electron-solid interaction. Effects such as sample charging and beam induced damage also, increasingly, influence performance and the choice of operating conditions.
TL;DR: The evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis is reviewed with reference to the role of microscopy and mechanisms by which lysOSomal lipid engorgement occurs are explored.
Abstract: Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.
TL;DR: A two- and three-dimensional fiber middle-line tracing algorithm is described that may be used to quantify collagen fibril organization and utilized computer simulation and statistical analysis to validate the developed algorithm.
Abstract: Recent evidence supports the notion that biological functions of extracellular matrix (ECM) are highly correlated to not only its composition but also its structure. This article integrates confocal microscopy imaging and image-processing techniques to analyze the microstructural properties of ECM. This report describes a two- and three-dimensional fiber middle-line tracing algorithm that may be used to quantify collagen fibril organization. We utilized computer simulation and statistical analysis to validate the developed algorithm. These algorithms were applied to confocal images of collagen gels made with reconstituted bovine collagen type I, to demonstrate the computation of orientations of individual fibers.
TL;DR: Using cytoskeleton stabilizing protocols on membrane-extracted parasites and novel imaging with high-resolution low-voltage field emission scanning electron microscopy (LVFESEM), a network of actin-sized filaments just below the cell membrane is visualize for the first time.
Abstract: The protozoan parasite Toxoplasma gondii is representative of a large group of parasites within the phylum Apicomplexa, which share a highly unusual motility system that is crucial for locomotion and active host cell invasion. Despite the importance of motility in the pathology of these unicellular organisms, the motor mechanisms for locomotion remain uncertain, largely because only limited data exist about composition and organization of the cytoskeleton. By using cytoskeleton stabilizing protocols on membrane-extracted parasites and novel imaging with high-resolution low-voltage field emission scanning electron microscopy (LVFESEM), we were able to visualize for the first time a network of actin-sized filaments just below the cell membrane. A complex cytoskeletal network remained after removing the actin-sized fibers with cytochalasin D, revealing longitudinally arranged, subpellicular microtubules and intermediate-sized fibers of 10 nm, which, in stereo images, are seen both above and below the microtubules. These approaches open new possibilities to characterize more fully the largely unexplored and unconventional cytoskeletal motility complex in apicomplexan parasites.
TL;DR: Cryo- and cryoetch-HRSEM provided vast vistas of hydrogels at low and intermediate magnifications whereas high magnification recordings and anaglyphs provided a three-dimensional prospective and measurements on a molecular level.
Abstract: The micro- and nanoarchitectures of water-swollen hydrogels were routinely analyzed in three dimensions at very high resolution by two cryopreparation methods that provide stable low-temperature specimens for in-lens high magnification recordings. Gemini surfactants (gS), poly-N-isopropylacrylamides (p-NIP Am), and elastin-mimetic di- (db-E) and triblock (tb-E) copolymer proteins that form hydrogels have been routinely analyzed to the sub-10-nm level in a single day. After they were quench or high pressure frozen, samples in bulk planchets were subsequently chromium coated and observed at low temperature in an in-lens field emission SEM. Pre-equilibrated planchets (4-40 degrees C) that hold 5-10 microl of hydrogel facilitate dynamic morphological studies above and below their transition temperatures. Rapidly frozen samples were fractured under liquid nitrogen, low-temperature metal coated, and observed in-lens to assess the dispersion characteristics of micelles and fragile colloidal assemblies within bulk frozen water. Utilizing the same planchet freezing system, the cryoetch-HRSEM technique removed bulk frozen water from the hydrogel matrix by low-temperature, high-vacuum sublimation. The remaining frozen solid-state sample faithfully represented the hydrogel matrix. Cryo- and cryoetch-HRSEM provided vast vistas of hydrogels at low and intermediate magnifications whereas high magnification recordings and anaglyphs (stereo images) provided a three-dimensional prospective and measurements on a molecular level.
TL;DR: In this paper, the authors used fluorescent speckle microscopy to evaluate the influence of microtubule dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation.
Abstract: We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 μm/s, too slow to drive chromatid-to-pole motion at 0.11 μm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.
TL;DR: It is found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host–pathogen systems and is highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.
Abstract: This article reports on the use of high pressure freezing followed by freeze substitution (HPF/FS) to study ultrastructural details of host–pathogen interactions in fungal diseases of plants. The specific host–pathogen systems discussed here include a powdery mildew infection of poinsettia and rust infections of daylily and Indian strawberry. The three pathogens considered here all attack the leaves of their hosts and produce specialized hyphal branches known as haustoria that invade individual host cells without killing them. We found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host–pathogen systems. Preservation of fungal and host cell membranes was particularly good and greatly facilitated the detailed study of host–pathogen interfaces. In some instances, HPF/FS provided information that was not available in samples prepared for study using conventional chemical fixation. On the other hand, we did encounter various problems associated with the use of HPF/FS. Examples included freeze damage of samples, inconsistency of fixation in different samples, separation of plant cell cytoplasm from cell walls, breakage of cell walls and membranes, and splitting of thin sections. However, we believe that the outstanding preservation of ultrastructural details afforded by HPF/FS significantly outweighs these problems and we highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.
TL;DR: The article compares the relative stability of MCM-41 and related mesoporous materials in electron beam at an accelerating voltage of 100–300 kV with similar research that has been carried out on nonporous and microporous silicates, especially α-quartz and zeolite Y.
Abstract: The article compares the relative stability of MCM-41 and related mesoporous materials in electron beam at an accelerating voltage of 100-300 kV. The work encountered in electron microscopy presents a comparison with similar research that has been carried out on nonporous and microporous silicates, especially alpha-quartz and zeolite Y. The trends in stability are analyzed, classifying the effects of sample preparation, organic and inorganic moieties, and electron accelerating voltage on beam stability. A higher synthesis temperature, the use of an acid catalyst in the synthesis, and the presence of additional organic or inorganic material within the channels were all found to stabilize these materials. The dose required to completely disrupt the structure increased with accelerating voltage for nearly all samples, suggesting a primarily radiolytic damage mechanism. The exception, MCM-41 containing nanometer-sized titania particles in its channels, was found to be almost insensitive to accelerating voltage.
TL;DR: The origins of ASTM date back to the end of the 19 century as mentioned in this paper, when the railroads linked the nation, and the steel purchased from one mill for a particular application could have greatly different properties than a similar alloy purchased from another mill.
Abstract: The origins of ASTM date back to the end of the 19 century. At this time two of the largest industries in America were the railroads and the steel industry. While the railroads linked the nation, the steel purchased from one mill for a particular application could have greatly different properties than a similar alloy purchased from another mill. Establishing voluntary, consensus, conformance standards in the steel industry was one of the driving forces for the creation of ASTM. With this in mind, it seems obvious that the first ASTM committee to be formed was A01, Steel. As standards for steel alloys were being developed, the next logical progression was the development of standard methodologies to test materials; hence, committee E01 on test methods was formed. In 1916 committee E04 was spun off from committee E01. While the first focus of E04 was to develop a set of standard magnifications to use for evaluating steel microstructures, with time the scope became much broader. The current name \"On Metallography\" was adopted in 1921. Today ASTM International has over 30,000 members from more than 100 countries, and is responsible for over 11,000 standards. Committee E04 is currently responsible for 36 standards concerning metallography and similarly related topics. Standard E384, Standard Test Method for Microindentation Hardness of Materials is one of the standards in Committee E04's charge [1].
TL;DR: To analyze the three-dimensional fine surface morphology of the copolymers, to the resolution level of a few nanometers, the method employed cryo-HRSEM and was able to visualize for characterization purposes the fine fibril networks formed from the micellar aggregates over the surface of the hydrogel.
Abstract: Elastin-mimetic block copolymers were produced by genetic engineering. Genetically driven synthesis permitted control of the final physiochemical characteristics of the block copolymers. We designed BB and BAB block copolymers in which the A-block was hydrophilic and the B-block was hydrophobic. By designing the copolymers in this manner, it was proposed that they would self-assemble into micellar aggregates that, at high concentration, would form thermoreversible hydrogels. To analyze the three-dimensional fine surface morphology of the copolymers, to the resolution level of a few nanometers, we employed cryo-HRSEM. This method provided vast expanses of the specimen in its frozen hydrated state for survey. In our initial cryo-HRSEM studies, we observed the protein filaments and micelles surrounded by lakes of vitreous ice. Upon examination at low and intermediate magnifications, there was an extensive honeycomb-like filamentous network. To delineate the fine morphology of the hydrogel network at high magnification and to greater depths, we cryoetched away unbound water from the sample surface, in high vacuum, prior to chromium deposition. By using this technique, we were able to visualize for characterization purposes the fine fibril networks formed from the micellar aggregates over the surface of the hydrogel.
TL;DR: The size of the focussed electron beam was found to have a considerable effect on the relative intensities of the CL emission peaks, and SEM parameters such as the objective lens aperture size, astigmatism, and the condenser lens setting must also be considered when assessing CL data based on intensity measurements from this material.
Abstract: Cathodoluminescence ~CL! spectra from silicon doped and undoped wurtzite n-type GaN have been measured in a SEM under a wide range of electron beam excitation conditions, which include accelerating voltage, beam current, magnification, beam diameter, and specimen temperature. The CL intensity dependence on excitation density was analyzed using a power-law model ~ICL @ J m ! for each of the observed CL bands in this material. The yellow luminescence band present in both silicon and undoped GaN exhibits a close to cube root ~m 5 0.33! dependence on electron beam excitation at both 77 K and 300 K. However, the blue ~at 300 K! and donor-acceptor pair ~at 77 K! emission peaks observed in undoped GaN follow power laws with exponents of m 5 1 and m 5 0.5, respectively. As expected from its excitonic character, the near band edge emission intensity depends linearly ~m 5 1! in silicon doped GaN and superlinearly ~m 5 1.2! in undoped GaN on the electron beam current. Results show that the intensities of the CL bands are highly dependent not only on the defect concentration but also on the electron-hole pair density and injection rate. Furthermore, the size of the focussed electron beam was found to have a considerable effect on the relative intensities of the CL emission peaks. Hence SEM parameters such as the objective lens aperture size, astigmatism, and the condenser lens setting must also be considered when assessing CL data based on intensity measurements from this material.
TL;DR: It is shown that nanometer-sized precipitates of atomic numbers higher than those of the surrounding crystalline matrix can be clearly revealed in a conventional transmission electron microscope by high-angle, centered dark-field imaging after minimizing the diffraction contrast.
Abstract: In this article, we show that nanometer-sized precipitates of atomic numbers higher than those of the surrounding crystalline matrix can be clearly revealed in a conventional transmission electron microscope by high-angle, centered dark-field imaging after minimizing the diffraction contrast. The effect is similar to that of Z-contrast STEM, albeit with a spatial resolution limited to 1 nm. Its sensitivity to atomic number differences between precipitates and matrix is about 10, which is demonstrated for precipitates formed after Er, Ge, Cr, and Si ion implantation into SiC.
TL;DR: It is suggested that the remaining fetal Leydig cells in the neonatal testis are associated with the involution or degeneration processes.
Abstract: The development of Leydig cells in mammals has been widely described as a biphasic pattern with two temporally mature Leydig cell populations, fetal stage followed by the adult generation beginning at puberty. In the present study, mouse Leydig cells were examined for apoptosis during postnatal testis development using electron microscopy and in situ DNA fragmentation by terminal deoxynucleotidyl transferase staining (TdT). Both the morphological study and the DNA fragmentation analysis showed that cellular death by apoptosis did not occur in Leydig cells during the neonatal, prepubertal, puberty, and adult periods. From these results, we suggest that the remaining fetal Leydig cells in the neonatal testis are associated with the involution or degeneration processes. In contrast, in the prepubertal and puberty stages, fragmentation of apoptotic DNA was detected in germ cells present in some seminiferous tubules.
TL;DR: High-precision measurements of pure element stable isotope pairs are presented that demonstrate mass has no influence on the backscattering of electrons at typical electron microprobe energies, and an improved backscatter (electron loss) factor based on a modified electron fraction average for the ZAF atomic number correction that provides a significant analytical improvement, especially where large atomic number corrections are required.
Abstract: We present high-precision measurements of pure element stable isotope pairs that demonstrate mass has no influence on the backscattering of electrons at typical electron microprobe energies. The traditional prediction of average backscatter intensities in compounds was pragmatically based on elemental mass fractions. Our isotopic measurements establish that this approximation has no physical basis. We propose an alternative model to mass fraction averaging, based on the number of electrons or protons, termed "electron fraction," which predicts backscatter yield better than mass fraction averaging. We also present an improved backscatter (electron loss) factor based on a modified electron fraction average for the ZAF atomic number correction that provides a significant analytical improvement, especially where large atomic number corrections are required.
TL;DR: The results suggest that the vitamin combination may find clinical use in the treatment of ovarian cancer and are consistent with cell death by autoschizis: decrease in cell size, cytoplasmic self-excisions, degradation of the nucleus and nucleolus without formation of apoptotic bodies and, ultimately, karyorrhexis and karyolysis.
Abstract: Human ovarian carcinoma cells (MDAH 2774) were treated with sodium ascorbate (VC), menadione (VK3) or with a VC:VK3 combination for I h and then studied using light microscopy (LM) and scanning (SEM) and transmission electron (TEM) microscopy. Plasma membrane damage (blisters and blebs, hairy aspect) results from vitamin C (VC) treatment, while cytoskeletal damage and self-morsellation are caused by vitamin K-3 (VK3) treatment. VC:VK3-treated cells exhibit exacerbated injuries characteristic of both VC and VK3 treatment as well as a significant decrease in cell diameters from 20-35 mum for control cells to 7-12 mum for VC:VK3 treatment. Moreover, after a 1-h exposure to the vitamin combination, autoschizis (43%), apoptosis (3%), and oncosis (1.9%) are observed at the percentages indicated. All cellular changes associated with autoschizis observed with SEM were confirmed by LM and TEM observations and are consistent with cell death by autoschizis: decrease in cell size, cytoplasmic self-excisions, degradation of the nucleus and nucleolus without formation of apoptotic bodies and, ultimately, karyorrhexis and karyolysis. These results also suggest that the vitamin combination may find clinical use in the treatment of ovarian cancer.
TL;DR: Second Harmonic Generation promises to be a potent new microscope imaging modality for the examination of structure and function in living cells.
Abstract: Second Harmonic Generation (SHG) has been developed in our laboratories as a highresolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison. In addition, we have developed molecular probes based on the styryl class of fluorescent chromophores that show large resonance-enhanced SHG signals when bound to membranes. The large charge redistributions between the ground and excited states in these dyes are the basis of the resonanceenhancement mechanism that enables SHIM of cell membranes. Figure 2 displays SHIM and TPEF micrographs of a neuronal cell stained with a styryl dye that has an appended chiral sugar moiety; we find that the covalent linkage of a chiral group to the chromophores can enhance the SHG signal by as much as a factor of 2. The styryl dyes have been developed as electrochromic indicators of membrane potential and have seen extensive use for spatiotemporal mapping of electrical activity by fluorescence microscope imaging. Interestingly, the SHG signals from these dyes show even greater sensitivity to potential than the fluorescence signal. Thus, SHIM promises to be a potent new microscope imaging modality for the examination of structure and function in living cells.